Quick and sensitive method of quantifying mycolic acid to develop anti-microbial agents and a diagnostic kit thereof

ABSTRACT

The present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.

FIELD OF THE INVENTION

[0001] The present invention relates to a rapid, sensitive, simple, andcost-effective spectrophotometric method of detecting and quantifyingmycolic acid in a mycolic acid-fuschin dye complex with absorbancemaxima ranging between 490-500 nm in the presence of various testcompounds, for screening mycolic acid biosynthesis inhibitors useful asanti-microbial agents and a diagnostic kit thereof comprising basicfuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml,phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v),and phenol and distilled water in the ratio ranging between 1:14 to1:25.

BACKGROUND OF THE INVENTION

[0002] Tuberculosis caused by Mycobacterium tuberculosis is a publichealth problem, which has increased in importance during the last twodecades, due in part to the increasing number of cases caused by theassociation of acquired immunodeficiency syndrome (AIDS) and theappearance of multiple drug-resistant strains. Other mycobacteria whichare often indistinguishable from tuberculosis have also increased.

[0003] The current method of detection is by determining the mycolicacid patterns from clinical isolates of sputum, cerebrospinal fluid,bronchial washing, corneal ulcer, and bone marrow, as well as fromacid-fast stain smear-positive clinical specimens. Standardized mycolicacid extraction methods are used to ensure the maximal extraction ofmycolic acid derivatives to enhance the sensitivity the method.Different chromatographic columns are used to identify the species ofmycolic acid. The immediate detection of bacterial containing mycolicacid is through acid fast staining and detection through the microscope.Lipid-rich cell walls are not permeable by ordinary stains.

[0004] The stain consisting of a basic dye (fuchsin) and phenol (a lipidsolvent) is used for the purpose. Phenol partially solubilizes cell walland allows fuchsin to penetrate the wall and bind to mycolic acid.

[0005] After fuchsin is incorporated, it is resistant to decolorizationeven after exposure to acid alcohol, a property that characterizesmycobacteria can be performed on unprocessed clinical specimens,concentrated specimens, or cultures.

[0006] This principle is widely used to identify bacteria producingmycolic acid with the help of a microscope. But an improved system basedon visual detection of the mycolic acid is the need of the hour toscreen out potential mycolic acid biosynthesis inhibiting compounds fromvast array of plant compounds.

[0007] The detection and quantification procedure can be made moreaccurate by using the help of spectrophotometer. The amount ofinhibition can be quantified simply through optical density measurementat a particular wavelength. This does not necessitate the complex, highcost technologies like IR, NMR, GC spectroscopy for the detection, whichare currently being used.

[0008] The amount of mycolic acid which, can not be quantified throughacid fast staining followed by microscopic detection can be quantifiedby systematic extraction and dye binding followed by spectrophotometricanalysis. Keeping this in mind a simple, rapid, cost effective procedurekit was devised to detect and quantify the mycolic acid which in turnleads to a high efficiency screen to identify potential inhibitors ofmycolic acid biosynthesis.

[0009] The protocols for extraction, isolation of mycolic acid anddetection bacteria producing mycolic acid through acid fast staining isavailable in the art. But the system as a whole is inefficient andrequires a microscope to detect the presence of the bacteria producingmycolic acid.

[0010] Similarly extraction, isolation and characterization of mycolicacid is carried out through different method of chromatography and IR,NMR spectroscopy. Our emphasis here was to generate a system to screen alarge amount of plant extracts and compounds with potential mycolic acidbiosynthesis inhibiting activity, rapidly and in low cost.

OBJECTS OF THE INVENTION

[0011] The main object of the present invention is to develop a quick,simple, sensitive, cost-effective method of identifying mycolic acid.

[0012] Another main object of the present invention is to develop aquick, simple, sensitive, cost-effective method of quantifying mycolicacid.

[0013] Further object of the present invention is to develop a quick,simple, sensitive, cost-effective spectrophotometric method ofidentifying and quantifying mycolic acid.

[0014] Yet another object of the present invention is to usecarbol-fuschin dye and mycolic acid complex to develop a quick, simple,sensitive, cost-effective spectrophotometric method of identifying andquantifying mycolic acid.

[0015] Still another object of the present invention is to develop amethod of screening test compounds for their mycolic acid inhibitoryproperty.

[0016] Still another object of the present invention is to develop adiagnostic kit for the detection of mycolic acid by spectrophotometrytechnique.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a rapid, sensitive, simple, andcost-effective spectrophotometric method of detecting and quantifyingmycolic acid in a mycolic acid-fuschin dye complex with absorbancemaxima ranging between 490-500 nm in the presence of various testcompounds, for screening mycolic acid biosynthesis inhibitors useful asanti-microbial agents and a diagnostic kit thereof comprising basicfuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml,phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v),and phenol and distilled water in the ratio ranging between 1:14 to1:25.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Accordingly, the present invention relates to a rapid, sensitive,simple, and cost-effective spectrophotometric method of detecting andquantifying mycolic acid in a mycolic acid-fuschin dye complex withabsorbance maxima ranging between 490-500 nm in the presence of varioustest compounds, for screening mycolic acid biosynthesis inhibitorsuseful as anti-microbial agents and a diagnostic kit thereof comprisingbasic fuschin dye in the concentration ranging between 0.1-1.0 gm/100ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1(v/v), and phenol and distilled water in the ratio ranging between 1:14to 1:25.

[0019] In an embodiment of the present invention a rapid, simple,sensitive, and cost effective method of screening mycolic acidbiosynthesis inhibitors useful as anti-microbial agents by quantifyingamount of mycolic acid produced by bacteria in presence and absence oftest compound or extract.

[0020] In another embodiment of the present invention, growing separatecultures of the said bacteria in the presence and absence of saidcompound or extract for time duration ranging between 40-60 hours.

[0021] In yet another embodiment of the present invention, lyophilizingbacterial pellet.

[0022] In still another embodiment of the present invention, extractingmycolic acid by conventional method.

[0023] In still another embodiment of the present invention, dissolvingextracted mycolic acid extract in hexane to obtain mycolic acidsolution.

[0024] In still another embodiment of the present invention, adding saidsolution to carbol-fuschin dye in the ratio ranging between 1:4 to 4:1(v/v).

[0025] In still another embodiment of the present invention, shakingproduct of step (e) vigorously to obtain a pink color mycolic acid-dyecomplex as an upper layer.

[0026] In still another embodiment of the present invention, quantifyingsaid mycolic acid spectrophotometrically at wavelength ranging between490-500 nm.

[0027] In still another embodiment of the present invention, determiningdegree of inhibition of mycolic acid biosynthesis in the compoundtreated bacterial culture.

[0028] In still another embodiment of the present invention, whereinquantifying mycolic acid level spectrophotometrically at wavelengthpreferably ranging between 494-496 nanometer.

[0029] In still another embodiment of the present invention, whereinratio of carbol Fuchsin dye to said extract is preferably in the ratio1:1 (v/v).

[0030] In still another embodiment of the present invention, whereincarbol fuschin is consisting of basic fuschin dye in the concentrationranging between 0.1-1.0 gm/100 ml, phenol, 95% ethanol, and distilledwater, said constituents in the ratio, phenol: ethanol in ratio rangingbetween 1:4 to 2:1 (v/v), and phenol: distilled water in the ratioranging between 1:25 to 1:14 (v/v).

[0031] In still another embodiment of the present invention, whereinsaid test compound or extract with inhibitory activity is added atconcentrations ranging between {fraction (1/25)} to ½ of minimuminhibitory concentration (MIC).

[0032] In still another embodiment of the present invention, whereinsaid anti-microbial agents are developed against microbes comprisingMycobacterium, Corynebacterium, and Nocardia.

[0033] In still another embodiment of the present invention, whereinsaid method is used to screen for inhibitors selected from a groupcomprising synthetic, semi-synthetic, natural compounds, and extracts.

[0034] In still another embodiment of the present invention, whereinintensity of color increases with increase in the concentration ofmycolic acid.

[0035] In still another embodiment of the present invention, whereinsaid method works for mycolic acid from all sources.

[0036] In a further embodiment of the present invention, a diagnostickit useful for identifying potential anti-microbial drugs againstmycolic acid producing microbes, said kit comprising carbol fuschin,methanol, toluene, hexane, concentrated sulphuric acid, bacterialpellet, and test compound or extract, with said constituents in theratio: methanol:toluene ranging between 1:3 to 3:1 (v/v),methanol:hexane ranging between 8:1 to 2:1 (v/v), concentrated sulphuricacid:hexane ranging between 1:8 to 1:2 (v/v), and Carbol fuschin:hexaneextract ranging between 1:4 to 4:1 (v/v), with methanol, toluene, andconcentrated sulphuric acid of above-mentioned ratio added intolyophilized bacterial pellet with final concentration of the sameranging between 0.1-3.0 gm/100 ml.

[0037] In still another embodiment of the present invention, whereincarbol fuschin is consisting of basic fuschin dye in the concentrationranging between 0.1-1.0 gm/100 ml, phenol, 95% ethanol, and distilledwater, said constituents in the ratio: phenol:ethanol in ratio rangingbetween 1:4 to 2:1 (v/v), and phenol:distilled water in the ratioranging between 1:14 to 1:25 (v/v).

[0038] In still another embodiment of the present invention, whereinsaid bacterial pellet is selected from a group comprising Mycobacterium,Corynebacterium, and Nocardia. In still another embodiment of thepresent invention, wherein said test compound or extract with inhibitoryactivity is added at concentrations ranging between {fraction (1/25)} to½ of minimum inhibitory concentration (MIC).

[0039] In another embodiment of the present invention Acid faststaining, a characteristic feature of the genus Mycobacteria causesmycolic acid to bind with carbol fuchsin dye to give the pink colour ofthe acid fast bacilli.

[0040] In yet another embodiment of the present invention, on the basisof this principle a spectrophotometric assay procedure was developed, inwhich the hexane extract of mycolic acid, when kept in a screw cap vialalong with carbol fuchsin dye, hexane fraction forms upper colorlesslayer and the dye forms the lower pink layer, but as the vial is mixedvigorously the carbol fuschin dye binds to mycolic acid present inhexane layer and forms an upper pink layer the intensity of whichincreases and a lower transparent layer in which intensity of pinkcolour decreases depending on the amount of mycolic acid present in theupper hexane layer.

[0041] In still another embodiment of the present invention, itindicates that during vigorous mixing of dye with hexane layer, it bindsto mycolic acid and form a complex and the amount of dye goes intohexane layer depending upon the concentration of mycolic acid in thehexane layer, whereas normal hexane without mycolic acid does not formthe complex to give the characteristic upper pink layer.

[0042] In still another embodiment of the present invention, Theinvention relates to a method for the screening of mycolic acidbiosynthesis inhibitors. More particularly the present invention relatesto a method to detect and quantify the relative amount of mycolic acidin a mixture or a solution or a culture leading to the identification ofinhibitors of mycolic acid biosynthesis, there by useful as a screeningprocedure to detect potential drug compounds for inhibition of mycolicacid biosynthesis. This invention also relates to a detection kit formycolic acid that can be used as a diagnostic kit. The present inventionhas direct implication in simplifying the procedure of rapid detectionof compounds inhibiting the biosynthesis of mycolic acid and ultimatelyleading to antimicrobial drug.

[0043] In still another embodiment of the present invention, the factthat mycolic acids in mycobacteria bind to carbol fuschin is well known.In fact, using this principal, the clinical specimens are routinelytested for the presence of mycobacterium in hospitals and diagnosticlabs but with the help of a microscope, which is a laborious procedure.Moreover, the microscope method can not be used for other applicationssuch as drug screening where quantification of mycolic acid productionby mycobacterium is a necessity.

[0044] In still another embodiment of the present invention, the methoddescribed in the instant Patent Application relies on the sameprinciples but enables us to quantify the mycolic acid production and/orits inhibition in mycobacterium. The instant Application is not soughtfor the principle but for the simple rapid, quantitative method for thedetection of mycolic acid using a diagnostic kit and technique ofspectrophotometry.

[0045] In still another embodiment of the present invention, the instantPatent Application facilitates an improved quantification of mycolicacid presence and thus the presence of mycobacterium in any givensample. Also it facilitates an improved quantification of inhibition ofmycolic acid synthesis useful in drug screening programmes for theidentification of mycolic acid inhibitors.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0046]FIG. 1 shows spectral scan of mycolic acid (5 micrograms/ml) inhexane.

[0047]FIG. 2 shows spectral scan of Carbol-fuschin dye.

[0048]FIG. 3 shows spectral scan of mycolic acid(5-micrograms/ml)-carbol fuschin complex in hexane.

[0049]FIG. 4 shows increase in absorbance with the increase inconcentration of mycolic acid when complexed with carbol fuschin dye.

[0050] The invention of this simple, novel and rapid system to detectpotential mycolic acid biosynthesis inhibitors has been illustrated inthe following examples. The said examples are provided to illustrate theinvention and therefore should not be considered to limit the scope ofthe present invention.

EXAMPLES Example 1 Mycolic Acid Extraction

[0051] Mycolic acids are a branched, β-hydroxy fatty acids which occurin the cell wall of Corynebacterium, Nocardia and Mycobacterium. Theirmolecular size varies with the genus, i.e. from 30, 50, 80-carbon atomsin the order described above. Because of the correlation between genusand mycolic acid size, the test for this biochemical marker is done inthe early stage of the identification of unknown actinomycetes. Themethod followed in this invention for the extraction of mycolic acid isaccording to Minniken et al (1975). After proper growth of the bacteria,the culture is centrifuged at 5000 RPM for 5 minutes and lyophilized.Dry bacteria (100 mg) are mixed with methanol (5 ml), Toluene (5 ml) andconcentrated sulphuric acid (0.2 ml) in a 20-ml screw cap tube (PolyTetra Fluoro Ethylene lined). The contents of the tube are mixedthoroughly and methanolysis is allowed to proceed for 12-16 hours at 75°C.(stationary incubation). The reaction mixture is allowed to cool andmycolic acid is extracted by adding 1 ml of Hexane. After vigorousmixing the mixture is allowed to settle and the upper hexane layercontaining mycolic acid is collected. Samples of the hexane extract werespotted on TLC coated with Merck Silica gel H (0.5 mm), along withstandard mycolic acid and the chromatogram is developed in petroleumether: Diethyl ether (85:15). The positions of the separated componentswere revealed by charring at 150-200° C. after spraying with chromicacid solution (5 gm K₂Cr₂O₇, in 5 ml H₂O, made up to 100 ml withconcentrated H2SO4, then diluted 10 times with water).

Example 2

[0052] The spectral scan of pure mycolic acid (procured from SigmaChemical Co.) was performed from 200 nm to 1000 nm. In visible rangeprominent peaks were not detected (FIG. 1).

Example 3

[0053] The spectral scan of carbol fuschin (Basic fuschin 0.3 g, Ethylalcohol (95%) 10.0 ml, Phenol 5.0 ml, Distilled water 95 ml) wasanalyzed from 200 nm to 1000 nm and a distinct peak at 538.71 wasrecorded in visible range indicating the colour of the stain (FIG. 2).

Example 4

[0054] When mycolic acid in hexane was added to carbol fuschin (1:1ratio v/v), carbol fuschin formed the lower pink layer and mycolic acidin hexane formed the upper transparent layer. After thorough mixing theupper hexane layer turned pink due to the complex formed between thestain and mycolic acid. Since mycolic acid is soluble in hexane and cannot come to the dense carbol fuschin layer the stain came to the upperhexane layer imparting pink colour to the upper layer. This simplesystem was exploited further to screen potential inhibitors of mycolicacid biosynthesis. The UV-Visible spectra of the complex of mycolic acidand fuschin in hexane layer was scanned from 200 nm to 1000 nm and founda shift in the peak of stain 538.71 nm to 495.89 nm indicating theformation of a complex with mycolic acid (FIG. 3).

Example 5

[0055] To confirm again that mycolic acid is the only candidateresponsible for formation of such complex a calorimetric assay of hexaneextract from Bacillus subtilis and Escherichia coli (non producer ofmycolic acid) were used and the observations are described in thisexample. When only hexane layer was mixed with carbol fuschin no changein colour observed. The upper hexane layer remained colorless and thelower dye layer pink, even after vigorous mixing. Similar observationswere recorded when hexane layer contained the extract of Bacillussubtilis and Escherichia coli. But the pink colour intensity of thelower layer carbol fuschin dye decreased when the hexane layer washaving the Mycobacterium smegmetis extract and broth with the upperhexane layer turning to pink with vigorous mixing indicating thepresence of mycolic acid. TABLE 1 Colour of carbol fuschin and hexanelayers in different treatments Colour of the Colour of the Contentslower layer Upper layer Dye (Carbol Fuchsin) + Hexane Pink Colorless Dye(Carbol Fuchsin) + Hexane Reduced intensity of Pink extract ofMycobacterium pink colour smegmatis Dye (Carbol Fuchin) + Hexane PinkColorless extract of Bacillus subtilis Dye (Carbol fuchsin) + HexanePink Colorless extract of Escherichia coli dye (Carbol fuchsin) + BrothReduced intensity of Pink Culture of Mycobacterium pink colour smegmatis

[0056] The following strains were used:

[0057] 1. M.smegmatis MTCC 6 (equivalent to ATCC 14468)=J.Gen.Microbiol.28: 339 (1962).

[0058] 2. B.subtilis MTCC 121 (equivalent to ATCC 6051)=Type strain,phage host, blood screening for phenylketonuria.

[0059] 3. E. coli MTCC 739 (equivalent to ATCC 10536)=Standardantibiotic test strain, J.Bacteriol. 54:549 (1947).

[0060] (MTCC=Microbial type culture collection, Institute of MicrobialTechnology, Chandigarh, India)

Example 6

[0061] The working of the system was characterized in details usingdifferent concentration of mycolic acid. Different concentrations (100,250, 500, 1000 μg/ml) of mycolic acid were allowed to form complex withthe stain as described in the previous sections and the increase inabsorbance patterns were recorded indicating the increase in theintensity of the colour (FIG. 4).

Example 7

[0062] By using this method we quantified the mycolic acid produced bythe control as well as treated cells of Msmegmatis with different plantextracts and compounds. Some of the compounds are known to be inhibitingmycolic acid biosynthesis and others are plant extracts and unknowncompounds. The compounds and the extracts were selected on the basis ofloosening of Acid Fast stain in Mycobacterium smegmatis.

[0063] The culture of Mycobacterium smegmatis was grown in NutrientBroth containing the sub lethal concentration (½ MIC) of thecompounds/Extracts for 48 hrs. The cells were harvested bycentrifugation and the pellets were lyophilized. Equal amount of thecells (100 mg) were taken both of control and treated cells and kept formethanolysis overnight as described in Example 1. Extracted hexane layerwere allowed to dry and a common stock (5 mg/ml) was used to run a TLCplate to determine how much the inhibition of Mycolic acid bycalculation the amount of produced Mycolic acid by Densitometer.

[0064] Compounds, which were showing loosening of Acid fast staining,were also tested for spectrophotometric assay. The strain ofMycobacterium smegmatis giving 100% Acidfast staining was grown for 48hours in 50 ml culture media. As described earlier treatment ofdifferent compounds were given at sub-lethal concentrations inreplicates were also grown for 48 hours. The control along withdifferent treated cells were pelleted by centrifugation and lyophilizedto get the dry pellet of the cells. Equal weight (100 mg) of the cells(control as well as treated) were kept for methanolysis overnight. After18-hrs incubation hexane fraction was extracted and dried. A commonstock (5 μg/ml) was made for the control as well as treated cells byusing hexane as a solvent. Equal amount (v/v) of carbol fuchsin dye wasadded and mixed properly, upper hexane layer which was transparentinitially, turned into a pink colour whereas in the case of treatedcells the intensity of the pink colour was less than the control cells.

[0065] The details of the treatments and the absorbance by differenttreatments have been described in Table 3. TABLE 2 Concentration of thecompound/ Stock used extract for to take absorbance atCompounds/extracts treatment the absorbance 495.89 nm Catharanthusroseus 312 μg/ml 5 mg/ml 0 root extract in ethanol Thebaine 150 μg/ml 5μg/ml 0 Isoniazid  1 μg/ml 5 μg/ml 0 Control — 5 μg/ml 1.4885

[0066] Isoniazid is a known mycolic acid biosynthesis inhibitor whichwhen treated with Mycobacterium smegmatis does not allow the productionof mycolic acid. Similarly the other plant based compounds thebaine andextracts like “Catharanthus roseus root ethanolic extract” do not allowthe synthesis of mycolic acid. So this method can be used to screen thesynthetic, semi-synthetic or natural compounds and the extracts may befrom prokaryotic or eukaryotic source. Similarly, the mycolic acidbiosynthesis inhibition can be checked not only for mycobacteria butalso in other groups like Corynebacterium and Nocardia.

[0067] The person skilled in the art may use different species ofmycolic acid and different dye complexing with mycolic acid producingdifferent absorption maximas but the essence of the invention is thatany dye complexing with different species of mycolic acid and producingdifferent absorption maxima can be assayed at that wavelength if theoptical density at that maxima vary proportionately with theconcentrations.

[0068] Above illustrations in the examples describes in details the useof already invented procedure to device novel, quick and low cost systemof screening the potential inhibitors of mycolic acid biosynthesis. Thissystem may look simple but has tremendous importance and potential asmillions of compounds and extracts are to be screened to arrive in apotential non-toxic drug formulation. The commercial potential of theinvention may also be exploited in form of a kit to detect mycolic acidthat will have an immediate impact in the diagnostic scene formycobacterium detection.

1. A rapid, simple, sensitive, and cost effective method of screeningmycolic acid biosynthesis inhibitors useful as anti-microbial agents byquantifying amount of mycolic acid produced by bacteria in presence andabsence of test compound or extract, said method comprising: a. growingseparate cultures of the said bacteria in the presence and absence ofsaid compound or extract for time duration ranging between 40-60 hours,b. lyophilizing bacterial pellet, c. extracting mycolic acid byconventional method, d. dissolving extracted mycolic acid extract inhexane to obtain mycolic acid solution, e. adding said solution tocarbol-fuschin dye in the ratio ranging between 1:4 to 4:1 (v/v), f.shaking product of step (e) vigorously to obtain a pink color mycolicacid-dye complex as an upper layer, g. quantifying said mycolic acidspectrophotometrically at wavelength ranging between 490-500 nm h.determining degree of inhibition of mycolic acid biosynthesis in thecompound treated bacterial culture.
 2. A method as claimed in claim 1,wherein quantifying mycolic acid level spectrophotometrically atwavelength preferably ranging between 494-496 nanometer
 3. A method asclaimed in claim 1, wherein ratio of carbol Fuchsin dye to said extractis preferably in the ratio 1:1 (v/v).
 4. A method as claimed in claim 1,wherein carbol fuschin is consisting of basic fuschin dye in theconcentration ranging between 0.1-1.0 gm/100 ml, phenol, 95% ethanol,and distilled water, said constituents in the ratio: (i) phenol: ethanolin ratio ranging between 1:4 to 2:1 (v/v), and (ii) phenol: distilledwater in the ratio ranging between 1:14 to 1:25 (v/v).
 5. A method asclaimed in claim 1, wherein said test compound or extract withinhibitory activity is added at concentrations ranging between {fraction(1/25)} to ½ of minimum inhibitory concentration (MIC).
 6. A method asclaimed in claim 1, wherein said anti-microbial agents are developedagainst microbes comprising Mycobacterium, Corynebacterium, andNocardia.
 7. A method as claimed in claim 1, wherein said method is usedto screen for inhibitors selected from a group comprising synthetic,semi-synthetic, natural compounds, and extracts.
 8. A method as claimedin claim 1, wherein intensity of color increases with increase in theconcentration of mycolic acid.
 9. A method as claimed in claim 1,wherein said method works for mycolic acid from all sources.
 10. Adiagnostic kit useful for identifying potential anti-microbial drugsagainst mycolic acid producing microbes, said kit comprising carbolfuschin, methanol, toluene, hexane, concentrated sulphuric acid,bacterial pellet, and test compound or extract, with said constituentsin the ratio: (i) methanol:toluene ranging between 1:3 to 3:1 (v/v),(ii) methanol:hexane ranging between 2:1 to 8:1 (v/v), (iii)concentrated sulphuric acid:hexane ranging between 1:2 to 1:8 (v/v), and(iv) Carbol fuschin:hexane extract ranging between 1:4 to 4:1 (v/v).with methanol, toluene, and concentrated sulphuric acid ofabove-mentioned ratio added into lyophilized bacterial pellet with finalconcentration of the same ranging between 0.1-3.0 gm/100 ml.
 11. The kitas claimed in claim 10, wherein carbol fuschin is consisting of basicfuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml,phenol, 95% ethanol, and distilled water, said constituents in theratio: (i) phenol:ethanol in ratio ranging between 1:4 to 2:1 (v/v), and(ii) phenol:distilled water in the ratio ranging between 1:14 to 1:25(v/v).
 12. The kit as claimed in claim 10, wherein said bacterial pelletis selected from a group comprising Mycobacterium, Corynebacterium, andNocardia.
 13. The kit as claimed in claim 10, wherein said test compoundor extract with inhibitory activity is added at concentrations rangingbetween {fraction (1/25)} to ½ of minimum inhibitory concentration(MIC).